The paracellular route is the dominant pathway for passive solute flow across the intestinal epithelial barrier, and its permeability depends on the regulation of the intercellular tight junction (tj), also known as the zonula occludens (ZO). The tj is a dynamic and complex structure whose physiological regulation remains largely undefined. During the 7 years of funding of this grant, our studies have focused on the mechanism(s) of action of a protein elaborated by Vibrio cholerae, zonula occludens toxin (Zot). Our experiments using Zot as a tool to gain insights into the regulation of tj function led to the discovery of zonulin, a human eukaryotic Zot analogue, and to the definition of some of its physiological (innate immune mucosal response of the gut) and pathological (autoimmune diseases) roles. We have demonstrated that Zot and zonulin each target the same surface intestinal receptor and activate the same intracellular signaling events that lead to reversible tj disassembly. We have extended our findings to disease states characterized by a leaky gut and have established the role of zonulin in their pathogenesis. We have applied genetic, biochemical, and physiologic techniques to define the structural and functional requirements for activation of the zonulin system. Our overall hypothesis is that the 12 kDa N-terminal portion of Zot and its mammalian analogue zonulin target the same specific receptor (the protease activated receptor (PAR)2) preferentially expressed on the surface of mature cells of the small intestine. This receptor-ligand interaction is coupled to activation of phospholipace C (PLC), protein kinase C (PKC)a, actin polymerization, and tj disassembly. We hypothesize that PKC-mediated phosphorylation of tj proteins alter protein-protein interaction within the tj multiprotein complex leading to the reversible tj disassembly. The long-term objective of this proposal is to use Zot as a probe to elucidate the role of the zonulin system in physiological and pathological states. We will focus on the structural requirements to activate the intracellular signaling involved in Zot/zonulin-mediated tj disassembly, and on establishing the Zot/zonulin-mediated activation of PAR-2 to gain insights into tj regulation at the cellular and molecular levels in health and disease.